Method for preparing cell cultures from biological specimens for chemotherapeutic and other assays

ABSTRACT

An improved method for preparing a cell culture is disclosed. The method includes culturing a multicellular tissue explant in the presence of growth medium that is substantially free of enzymes capable of digesting the explant and, subsequently, removing the explant at a predetermined time.

REFERENCE TO RELATED APPLICATIONS

[0001] Reference is made to U.S. Ser. No. 09/040,161, filed Mar. 17,1998, which is a continuation of U.S. Ser. No. 08/679,056, filed Jul.12, 1996, now U.S. Pat. No. 5,728,541. The disclosure of each of theforegoing of which is incorporated by reference herein.

FIELD OF THE INVENTION

[0002] The invention relates to methods for the preparation of a cellculture monolayer, and more particularly to methods for the preparationof a tumor cell culture monolayer that substantially comprises tumorcells.

BACKGROUND

[0003] Prior to approval for medical use in the United States, allpharmaceutical agents are subjected to rigorous testing for efficacy andsafety. Typically, methods of assessing the efficacy of a pharmaceuticalagent include complex studies of pooled patient samples or pooled data,and statistical interpretation of the results. The conclusions thatfollow such studies are inherently generalized or averaged over thesubject patient population. With pharmaceutical agents, however, andparticularly with cancer chemotherapeutic agents, the efficacy of anagent in treating an individual patient can vary greatly from thegeneralized data, often to the detriment of the individual patient. Theneed has long been recognized for a method of assessing the therapeuticpotential of pharmaceutical agents, including but not limited tochemotherapeutic agents, for their specific efficacy in an individualpatient.

[0004] Assays exist which expose malignant tissue of various types topharmaceutically-active agents for the purpose of assessing the bestchoice for therapeutic administration. For example, in Kruczynski, A.,et al., “Evidence of a direct relationship between the increase in thein vitro passage number of human non-small-cell lung cancerprimocultures and their chemosensitivity,” Anticancer Research, vol. 13,no. 2, pp. 507-513 (1993), chemosensitivity of non-small-cell lungcancers was investigated in in vivo grafts, in in vitro primocultures,and in commercially available cancer cell lines. The increase inchemosensitivity was documented and correlated with morphologicalchanges in the cells in question. Often, animal model malignant cellsand/or established cell cultures are tested with prospective therapyagents, see for example Arnold, J. T., “Evaluation of chemopreventiveagents in different mechanistic classes using a rat tracheal epithelialcell culture transformation assay,” Cancer Res., vol. 55, no. 3, pp.537-543 (1995).

[0005] According to prior art methods of using specific patient tumorcells to form an in vitro assay particular to that patient, the cellsare harvested (biopsied) and trypsinized (connective tissue digestedwith the enzyme trypsin) to yield a cell suspension suitable forconversion to the desired tissue culture form. The in vitro tissueculture cell preparations which result from these prior art methodstypically fail to accurately replicate the chemosensitivity of theoriginal tumor or other cell biopsy. This inability arises, in part,because the heterogeneity of cell population in the tumor tissue hasbeen disturbed in culture, or entirely obliterated such that the cellculture preparation is essentially monoclonal. Moreover, prior artmethods of culturing actual patient tissue samples inevitably result incell cultures with a significant level of nontarget cells such asfibroblasts or other stromal cells, which have a tendency to outgrow thetarget tumor cells in a cell culture. Furthermore, standard cloning andtissue culture techniques are complicated and expensive for use inindividualized patient testing. Thus, a need remains for improved andefficient methods of cell culture preparation that provide aheterogeneous cell population that substantially comprises target cellsfrom a particular patient. Such a cell culture preparation is useful indrug or chemotherapeutic agent screening to provide informationindicative of the in vivo reactivity of the cells, and thus the specificefficacy as to a particular patient.

SUMMARY OF THE INVENTION

[0006] The present invention provides methods for preparing a cellculture from a multicellular tissue extract. Cell cultures of theinvention have the advantage of closely resembling the in vivo cellpopulation from which they were obtained, thus providing an accurate andreliable proxy for the cell population in vivo. For example, a tumorcell culture of the invention comprises a population of cells thatmimics the tumor cell population in the patient from whom a tissueexplant is obtained. This allows chemosensitivity and chemoresistivitytesting that is highly-reliable in predicting the effects of therapeuticagents on the tumor in vivo. The invention is based, in part, on theinsight that timely removal of a cellular explant from culture resultsin a culture that is highly indicative of in vivo cell population. Theinvention provides further benefits recognized by culturing tissueexplants in a growth medium that is essentially free of digestiveenzymes.

[0007] Accordingly, in one aspect, the invention provides a cell culturesystem in which a multicellular tissue explant is placed in a growthmedium and is removed from the growth medium at a predetermined time.The explant is removed prior to the emergence from the explant of asubstantial number of non-target cells, resulting in a monolayer ofcells that is enriched for the cell population of interest. For example,it has been discovered that cells emerge as a monolayer from a culturedtumor tissue explant in an orderly fashion, the tumor cells emergingfirst, followed by stromal cell populations. If the tumor cell explantremains in culture, the stromal cells have been found to dominate thetumor cells in culture. This creates a culture that is enriched fromnon-target stromal cells and that is not reflective of the in vivo cellpopulation. Thus, in a tumor cell culture, the explant is removed fromthe growth medium prior to the emergence of a substantial number ofstromal cells from the explant. This provides a cell culture monolayerthat is predominantly composed of tumor cells. It has also beendiscovered that the cell cultures described above produce optimalresults in cell culture medium that is essentially free of digestiveenzymes.

[0008] The time at which an explant is removed from its culture mediumdepends upon the type of cells being cultured, the rate of emergence ofvarious cell types, and the desired purity of the resulting cell culturemonolayer. This can be determined empirically for a given cell type. Inthe case of tumor cells, the multicellular tissue explant is preferablyremoved when the cell culture monolayer is at about 10 to about 50percent confluency. In a preferred embodiment, the multicellular tissueexplant is removed at about 15 to about 25 percent confluency. In aparticularly preferred embodiment, the explant is removed at about 20percent confluency.

[0009] The invention further comprises the preparation of a cellsuspension from the cell culture monolayer. A tissue explant is culturedin an appropriate medium and is removed at a predetermined time,resulting in a monolayer enriched for the cells of interest. Asuspension is then made from the monolayer and cells of the suspensionare inoculated into at least one segregated site. In one embodiment, achemosensitivity assay is performed on the inoculated cell suspension byexposing the segregated site to at least one agent and assessing thechemosensitivity of the cells in the segregated site. Chemoresistivityassays are similarly performed.

[0010] In another embodiment, the invention provides methods fordetermining the chemosensitivity of a tissue in a patient by determiningthe chemosensitivity of a cell culture preparation from the patient. Inyet another embodiment, the invention provides methods for identifyingan agent having anti-tumorogenic effect in a patient by assessing thechemosensitivity of segregated sites of cells from a tumor cell cultureprepared according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention provides methods for preparing a cellculture monolayer by culturing a tissue sample from a patient.Ultimately, the culture may be used to screen at least one candidatetherapeutic or chemotherapeutic agent for efficacy as to a specificpatient, in which a tissue sample from the patient is harvested andseparately exposed to a plurality of treatments and/or therapeuticagents for the purpose of objectively identifying the chemosensitivityor chemoresistivity of the tissue sample and the best treatment or agentfor the patient. Tissue sample preparation techniques render this methodpractically as well as theoretically useful. According to the invention,the initial cohesive multicellular particulates (explants) of the tissuesample are prepared mechanically, rather than enzymatically, for initialtissue culture monolayer preparation. The multicellular tissue explantis removed from the culture growth medium at a predetermined time toboth allow for the growth of target cells and prevent substantial growthof non-target cells such as fibroblasts or stromal cells.

[0012] An important application of the present invention is thescreening of chemotherapeutic agents and other antineoplastic oranti-tumorogenic therapies against tissue culture preparations oftumorogenic cells from the patient from whom the sample is biopsied.Related anti-cancer therapies which can be screened using the methods ofthe invention are both radiation therapy and agents which enhance thecytotoxicity of radiation, as well as immunotherapeutic anti-canceragents. Screening processes for treatment or therapeutic agents fornonmalignant syndromes are also embraced within this invention, however,and include without limitation, agents which combat hyperproliferativediseases, such as psoriasis, or wound healing agents. Nor is the presentefficacy assay limited only to the screening of active agents whichspeed up (healing) or slow down (anti-cancer, anti-hyperproliferative)cell growth because agents intended to enhance or to subdueintracellular biochemical functions may be tested in the present tissueculture system as well. For example, the formation or blocking ofenzymes, neurotransmitters and other biochemicals may be screened withthe present assay methods prior to treatment of the patient.

[0013] By way of example, in one embodiment of the invention, a cellculture monolayer in accordance with the invention is prepared using thefollowing procedure. Many aspects of the following procedure may bealtered as necessary and as well known in the art. A biopsy ofnormecrotic, non-contaminated tissue is harvested from the patient byany suitable biopsy or surgical procedure known in the art. In apreferred embodiment, the tissue sample is tumor tissue. In oneembodiment, the biopsy is at least about 100 mg. Biopsy samplepreparation generally proceeds under sterile conditions. Cohesivemulticellular particulates (explants) are prepared from the tissuesample using mechanical fragmentation. In one embodiment, thismechanical fragmentation of the explant occurs in a medium substantiallyfree of enzymes that are capable of digesting the explant. In apreferred embodiment, the tissue sample is minced with sterile scissorsto prepare the explants. In a particularly preferred embodiment, thetissue sample is systematically minced by using two sterile scalpels ina scissor-like motion, or mechanically equivalent manual or automatedopposing incisor blades. This cross-cutting motion creates smooth cutedges on the resulting tissue multicellular particulates. In oneembodiment, multicellular particulates measuring about 1 mm³ may beproduced. After the tissue sample has been minced, the particles areplated in culture flasks (for example, 9 explants per T-25 or 20particulates per T-75 flask). The explants may be evenly distributedacross the bottom surface of the flask, followed by initial inversionfor about 10-15 minutes. The flask may then be placed in a non-invertedposition in a 37° C. CO₂ incubator for about 5-10 minutes. In anotherembodiment in which the tissue sample comprises brain cells, the flasksare placed in a 35° C., non-CO₂ incubator. Flasks should be checkedregularly for growth and contamination. According to a preferredembodiment of the invention, the multicellular explant is removed fromthe cell culture at a predetermined time, as described below. Over aperiod of a few weeks a monolayer will be produced. With respect to theculturing of tumor cells, it is believed (without any intention of beingbound by the theory) that tumor cells grow out from the multicellularexplant prior to stromal cells. Thus, by initially maintaining thetissue cells within the explant and removing the explant at apredetermined time, growth of the tumor cells (as opposed to stromalcells) into a monolayer is facilitated.

[0014] The use of the above procedure to form a cell culture monolayerculture maximizes the growth of tumor cells from the tissue sample, andthus optimizes ensuing tissue culture assay of various agents (e.g.,chemotherapeutic agents) to be tested. Once a primary culture and itsderived secondary monolayer tissue culture has been initiated, thegrowth of the cells may be monitored to oversee growth of the monolayerand ascertain the time to initiate the chemotherapy assay and todetermine the growth rate of the cultured cells. Prior to thechemotherapy assay, monitoring of the growth of cells may be conductedby visual monitoring of the flasks on a periodic basis, without killingor staining the cells and without removing any cells from the cultureflask. Data from periodic counting is then used to determine growthrates which may or may not be considered parallel to growth rates of thesame cells in vivo in the patient. If growth rate cycles can bedocumented, for example, then dosing of certain active agents can becustomized for the patient. The same growth rate can be used to evaluateradiation treatment periodicity, as well. It should be noted that withthe growth rate determinations conducted while the monolayers grow intheir flasks, the present method requires no hemocytometry, flowcytometry or use of microscope slides and staining, with all theirconcomitant labor and cost.

[0015] Monolayer growth rate may be monitored using, for example, aphase-contrast inverted microscope. In one embodiment, culture flasksare incubated in a (5% CO₂) incubator at about 37° C. The flask isplaced under the phase-contrast inverted microscope, and ten fields(areas on a grid inherent to the flask) are examined using the 10×objective. In general, the ten fields should be non-contiguous, orsignificantly removed from one another, so that the ten fields are arepresentative sampling of the whole flask. Percentage cell occupancyfor each field examined is noted, and averaging of these percentagesthen provides an estimate of overall percent confluency in the cellculture. When patient samples have been divided between two or amongthree or more flasks, an average cell count for the total patient sampleshould be calculated. The calculated average percent confluency shouldbe entered into a process log to enable compilation of data—and plottingof growth curves—over time. Alternatively, confluency may be judgedindependently for each flask. Monolayer cultures may be photographed todocument cell morphology and culture growth patterns. The applicableformula is:${{Percent}\quad {confluency}} = \frac{{estimate}\quad {of}\quad {the}\quad {area}\quad {occupied}\quad {by}\quad {cells}}{{total}\quad {area}\quad {in}\quad {an}\quad {observed}\quad {field}}$

[0016] Percent confluency=estimate of the area occupied by cells totalarea in an observed field

[0017] As an example, therefore, if the estimate of area occupied by thecells is 30% and the total area of the field is 100%, percent confluencyis 30/100, or 30%.

[0018] Following initial culturing of the multicellular tissue explant,the tissue explant is removed from the growth medium at a predeterminedtime. In one embodiment, the explant is removed from the growth mediumprior to the emergence of a substantial number of stromal cells from theexplant. Alternatively, the explant may be removed according to thepercent confluency of the cell culture. In one embodiment of theinvention, the explant is removed at about 10 to about 50 percentconfluency. In a preferred embodiment of the invention, the explant isremoved at about 15 to about 25 percent confluency. In a particularlypreferred embodiment, the explant is removed at about 20 percentconfluency. By removing the explant in either of the above manners, acell culture monolayer predominantly composed of target cells (e.g.,tumor cells) is produced. In turn, a substantial number of non-targetcells, such as fibroblasts or other stromal cells, fail to grow withinthe culture. Ultimately, this method of culturing a multicellular tissueexplant and subsequently removing the explant at a predetermined timeallows for increased efficiency in both the preparation of cell culturesand subsequent assays of various agents using the cultures. Adaptationof the above protocol for non-tumor cells is straightforward andgenerally constitutes an equivalent procedure.

[0019] The essence of the invention thus includes the important featureof the simplicity of the present system—cohesive multicellular explantsof the patient tissue to be tested are used to form cell monolayers;growth of those monolayers may be monitored for accurate prediction ofcorrelating growth of the same cells in vivo; explants are removed fromthe growth medium at a predetermined time, and differing concentrationsof a number of active agents may be tested for the purpose ofdetermining chemosensitivity of the tissue sample and the mostappropriate agent and concentration of that agent for actual patientexposure (according to the calculated cell growth rates). It is alsoimportant to note, in the context of the invention, that the presentsystem allows in vitro tests to be conducted in suspensions of tissueculture monolayers grown in nutrient medium under fast conditions (amatter of weeks), rather than with single cell progeny produced bydilution cloning over long periods of time. In some cases, the presentinvention provides a cell culture for a two stage assay for bothcytotoxicity and the longer-term growth inhibitory.

EXAMPLE Chemosensitivity Assay

[0020] Methods of the invention include methods for determining theefficacy of an active agent. The performance of the chemosensitivityassay used for screening purposes depends on the ability to deliver areproducible cell number to each row in a plate and/or a series ofplates, as well as the ability to achieve an even distribution of cellsthroughout a given well. The following exemplary procedure assures thatcells are reproducibly transferred from flask to microtiter plates, andcells are evenly distributed across the surface of each well.

[0021] An initial step in preparing the microtiter plates is preparingand monitoring the monolayer as described above with the removal of theexplant at 20 percent confluency. The following example shows anexemplary protocol which is susceptible of variation as will be apparentto one skilled in the art. Cells were removed from the culture flask anda cell pellet was prepared by centrifugation. The cell pellet derivedfrom the monolayer was then suspended in 5 ml of the growth medium,mixed in a conical tube and subsequently rocked back and forth 10 times.A 30 μl droplet from the center of the conical tube was pipetted intoone well of a 96 well plate. A fresh pipette was then used to pipette a30 μl aliquot of trypan blue solution, which was added to the same well,and the two droplets were mixed with repeated pipette aspiration. Theresulting admixture was then applied to a hemocytometer chamber forexamination using a standard light microscope. Cells were counted in allof four hemocytometer quadrants, under 10× magnification. Only thosecells which had not taken up the trypan blue dye were counted. Usingmeans known in the art, the quadrant count values were checked, logged,multiplied by 10⁴ to give cells/ml, and the total amount of fluid(growth medium) necessary to suspend remaining cell aliquots wascalculated accordingly.

[0022] After the desired concentration of cells in medium has beendetermined, additional cell aliquots from the monolayer were suspendedin growth medium via rocking and then, loaded into a Terasaki dispenser.Aliquots of the prepared cell suspension were delivered into themicrotiter plates using Terasaki dispenser techniques known in the art.Alternatively, an electronic multichannel pipettor commerciallyavailable from Matrix Technology Corp. may be used. A plurality ofplates may be prepared from a single cell suspension as needed. Plateswere subsequently incubated in an incubator box by means known in theart. Upon preparation of the cell suspension, cells from the suspensionmay be inoculated into segregated sites for subsequent assays. At leastone agent may be exposed to the segregated sites to determine thechemosensitivity of the tissue samples, as well as the therapeutic orchemotherapeutic effects of the agents on the tissue sample.

[0023] The following example provides an exemplary protocol for assayingactive agents in accordance with the invention. During this portion ofthe inventive assay, the appropriate amount of specific active agent wastransferred into the microtiter plates prepared as described above. Ageneral protocol, which may be adapted, follows. Each microtiter platewas microscopically examined for cell adhesion. Control solution wasdispensed into delineated rows of wells within the grid in themicrotiter plate, and appropriate aliquots of active agent to be testedwere added to the remaining wells in the remaining rows. Ordinarily,sequentially increasing concentrations of the active agent being testedwere administered into progressively higher numbered rows in the plate.The plates were then incubated in an incubator box at 37° C. under 5%CO₂. After a predefined exposure time, the plates were blotted withsterile gauze to remove the agent, washed with Hank's Balance SaltSolution, flooded with growth medium, and replaced in the incubator inan incubator box for a predefined time period, after which the plateswere fixed and stained for evaluation.

[0024] Fixing and staining may be conducted according to a number ofsuitable procedures; the following is representative. After removal ofthe plates from the incubator box, culture medium were poured off andthe plates were flooded with Hank's Balance Salt Solution. Afterrepeated flooding (with agitation each time) the plates were thenflooded with reagent grade ethanol for 2-5 minutes. The ethanol was thenpoured off. Staining was accomplished using a DAPI (4′,6-diamidino-2-phenylindole, dilactate) staining method. Each plate wasflooded with a DAPI/water solution, with a concentration of about 400nM, and allowed to stand for at least 10 minutes, after which theDAPI/water was poured into a beaker. The plates were then dipped into abeaker of running water to remove the excess DAPI. Cells per well maythen be counted manually or by automated and/or computerized means, toderive data regarding chemosensitivity of cells at variousconcentrations of exposure. One particularly useful computer operatingenvironment for counting cells is the commercially available ZeissAxiovert S100 Automatic Inverted Flourescence Microscope and Computer.

[0025] The above procedures do not change appreciably when cell growthpromoters are assayed rather than cell arresting agents such aschemotherapeutic agents. The present assay allows cell death or cellgrowth to be monitored with equal ease. In any case, optimization of useof the present system will involve the comparative testing of a varietyof candidate active agents, for selection of the best candidate forpatient treatment based upon the in vitro results. One particularlyadvantageous embodiment of the above-described invention comprises atwo-stage assay for cytotoxicity followed by evaluation of longer-terminhibitory effect chemotherapeutic agents may thus be evaluatedseparately for both their direct chemotherapeutic effect as well as fortheir longer duration efficacy.

[0026] The invention is not to be limited only to the illustrativedescription provided herein. Variations, modifications, and otherimplementations of what is described herein will occur to those ofordinary skill without departing from the spirit and scope of theinvention.

What is claimed is:
 1. A method for preparing a cell culture, the methodcomprising: culturing a multicellular tissue explant in the presence ofgrowth medium that is substantially free of enzymes capable of digestingsaid explant; and removing said multicellular tissue explant from saidgrowth medium at a predetermined time, thereby to produce a cell culturemonolayer.
 2. The method of claim 1, further comprising preparing a cellsuspension from said cell culture monolayer.
 3. The method of claim 1,further comprising inoculating cells from said cell culture monolayerinto at least one segregated site.
 4. The method of claim 1, whereinsaid tissue explant comprises tumor tissue.
 5. The method of claim 1,further comprising the step of mechanically fragmenting themulticellular tissue explant in a medium that is free of enzymes thatare capable of digesting said explant.
 6. The method of claim 1,comprising the step of removing said multicellular tissue explant fromsaid growth medium prior to an emergence of a substantial number ofstromal cells from said explant, thereby to produce a cell culturemonolayer that is predominantly composed of non-stromal cells.
 7. Themethod of claim 6 wherein said non-stromal cells comprise tumor cells.8. The method of claim 1, wherein the cell culture monolayer exhibits apercent confluency, and wherein the step of removing said multicellulartissue explant comprises removing said multicellular tissue explant atabout 10 to about 50 percent confluency.
 9. The method of claim 8,wherein the step of removing said multicellular tissue explant comprisesremoving said multicellular tissue explant at about 15 to about 25percent confluency.
 10. The method of claim 8, wherein the step ofremoving said multicellular tissue explant comprises removing saidmulticellular tissue explant at about 20 percent confluency.
 11. A cellculture preparation resulting from the method of claim 1 or
 10. 12. Amethod for determining the chemosensitivity of a tissue in a patient toan agent, the method comprising the steps of: (a) culturing amulticellular tissue explant from a patient in the presence of growthmedium that is substantially free of enzymes capable of digesting saidexplant so as to produce a cell culture monolayer; (b) removing saidmulticellular tissue explant from said growth medium at a predeterminedtime; (d) inoculating cells from said cell culture monolayer into atleast one segregated site; (e) exposing said segregated site to at leastone agent; and (f) assessing the chemosensitivity of said cells in saidsegregated site, wherein said chemosensitivity of said cells isindicative of the chemosensitivity of the tissue in the patient.
 13. Themethod of claim 12, wherein said multicellular tissue explant comprisestumor tissue.
 14. The method of claim 13, wherein the cell culturemonolayer exhibits a percent confluency, and wherein the step ofremoving said multicellular tissue explant comprises removing saidmulticellular tissue explant at about 10 to about 50 percent confluency.15. The method of claim 14, wherein the step of removing saidmulticellular tissue explant comprises removing said multicellulartissue explant at about 15 to about 25 percent confluency.
 16. Themethod of claim 14, wherein the step of removing said multicellulartissue explant comprises removing said multicellular tissue explant atabout 20 percent confluency.
 17. A method for determining the efficacyof an agent, the method comprising the steps of: (a) preparing a cellculture in accordance with claim 3; (b) exposing said segregated site toat least one agent; and (c) assessing the chemosensitivity of said cellsin said segregated site, thereby to determine the efficacy of saidagent.
 18. The method of claim 17, wherein the cell culture monolayerexhibits a percent confluency, and wherein the step of removing saidmulticellular tissue explant comprises removing said multicellulartissue explant at about 15 to about 25 percent confluency.
 19. Themethod of claim 18, wherein the step of removing said multicellulartissue explant comprises removing said multicellular tissue explant atabout 20 percent confluency.
 20. A method for identifying an agenthaving anti-tumorogenic effect, the method comprising the steps of: (a)preparing a cell culture in accordance with claim 4; (b) inoculatingcells from said cell culture monolayer into a plurality of segregatedsites (c) exposing each of said segregated sites to an agent; and (d)assessing the chemosensitivity of said cells in each of said segregatedsites, thereby to identify an agent having an anti-tumorogenic effect.